J. Anim Sci. 2008. 86:960-966. doi:10.2527/jas.2007-0549
© 2008 American Society of Animal Science
Caspase 3 is not likely involved in the postmortem tenderization of beef muscle1
K. R. Underwood,
W. J. Means and
M. Du2
Department of Animal Science, University of Wyoming, Laramie, WY 82071
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Abstract
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Postmortem proteolysis is an important determinant of beef tenderness. Caspase 3 is a protease that functions in apoptosis and has been shown to degrade myofibrillar proteins. Our objective was to evaluate whether caspase 3 activity is related to beef tenderness and muscle growth, and whether caspase 3 is activated in postmortem beef muscle. In experiment 1, longissimus thoracis (LT) and sternomandibularis muscle samples were obtained at 0, 0.25, 1, 3, 24, 72, and 240 h postmortem from 5 steers. In experiment 2, a group of 40 beef cattle was slaughtered at the University of Wyoming Meat Lab with 10 steers of different tenderness and growth characteristics chosen for the analysis of caspase 3 activity in the LT. In experiment 3, 10 steers with different tenderness but matched growth characteristics were chosen for analyses. In experiment 1, no significant activation (P = 0.70) of caspase 3 activity was detected; only a decreased activity at 72 (P = 0.05) and 240 h (P = 0.02) postmortem was observed. Western blot analysis of both muscle samples showed only the pro-caspase 3 form and failed to detect the activated enzyme. In experiment 2, caspase 3 activity in the LT immediately postmortem was greater (P = 0.05) for the cattle with increased Warner-Bratzler shear force values. No difference in caspase 3 activity was detected for experiment 3. Our results demonstrate that caspase 3 activity is not activated, with its activity decreasing with time postmortem, and caspase 3 activity is not associated with Warner-Bratzler shear force at slaughter. Therefore, caspase 3 is not anticipated to be involved in postmortem tenderization of beef.
Key Words: beef • caspase 3 • postmortem • proteolysis • tenderness
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INTRODUCTION
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Postmortem storage of muscle foods is known to increase tenderness subjectively and objectively (Koohmaraie et al., 1988
, 1991). Increased tenderness during postmortem storage has been attributed to proteolysis of myofibrillar proteins (Goll et al., 1983; Koohmaraie et al., 1988; Sentandreu et al., 2002). Researchers have attributed postmortem proteolysis to catheptic enzymes and the calpain/calpastatin system (Etherington et al., 1987; Koohmaraie et al., 1991). The calpain/calpastatin system has received much attention and is credited as a more feasible proteolytic system responsible for postmortem degradation of myofibrillar proteins (Koohmaraie et al., 1991; Uytterhaegen et al., 1994; Goll et al., 2003).
Recently, other proteolytic systems have been proposed to be active in postmortem muscle and affecting myofibrillar degradation (Herrera-Mendez et al., 2006; Kemp et al., 2006a). It has been suggested the ubiquitin proteasome system and caspases could play a role in postmortem myofibrillar degradation (Sentandreu et al., 2002; Herrera-Mendez et al., 2006; Kemp et al., 2006b). This hypothesis is based on the finding that these endogenous peptidases are capable of hydrolyzing myofibrillar proteins (Condorelli et al., 2001; Lee et al., 2004). Furthermore, Sentandreu et al. (2002) and Herrera-Mendez et al. (2006) proposed a mechanism by which hypoxic conditions in postmortem muscle could cause apoptosis, triggering caspase 3 activation. Up to now, the possible involvement of caspase 3 in the postmortem tenderization of beef muscle has not been evaluated. Hence, the objective of this study was to evaluate whether caspase 3 is involved in postmortem tenderization of beef muscle.
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MATERIALS AND METHODS
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All animal procedures were approved by the University of Wyoming Animal Care and Use Committee.
Muscle Sample Preparation
Experiment 1.
Five steers were obtained from the Fort Keogh USDA Research Station and were slaughtered at University of Wyoming Meat Laboratory. Longissimus thoracis (LT) muscle and sternomandibularis muscle samples (3 g) were excised at 0 (immediately after exsanguination), 0.25, 1, 3, 24, 72, and 240 h after slaughter. After removing all visible fat and connective tissue, muscle samples were snap-frozen in liquid nitrogen. Muscle samples were then stored at –80°C until caspase 3 activity and Western blotting analysis could be performed.
Experiment 2.
Forty British x continental cross-bred steers that had the same genetic background, age, and nutritional management were slaughtered in the University of Wyoming Meat Laboratory as described previously by Underwood et al. (2007b). The LT (2 g) was removed from the right side of carcasses within 10 min postmortem, trimmed free of all visible adipose tissue, and snap-frozen immediately in liquid nitrogen. Muscle samples were stored at –80°C until analysis was performed. Carcass data, Warner-Bratzler shear force analysis, estimates of muscle growth, and carcass composition were analyzed, as described previously by Underwood et al. (2007a). Briefly, LT area was determined according to the USDA (1997) at 48 h postmortem. The whole LT and semitendinosus muscle were dissected from the left side of the carcass at 7 d postmortem and weights were recorded. Carcass composition was estimated using a 9-10-11th-rib dissection, according to Hankins and Howe (1946).
Five crossbred steers with low Warner-Bratzler shear force (3.77 ± 0.20 kg) and 5 steers with high Warner-Bratzler shear force (4.44 ± 0.18 kg) were chosen for the analyses of caspase 3 activity.
Experiment 3.
Five crossbred steers with the lowest Warner-Bratzler shear force (3.60 ± 0.12 kg) and 5 steers with greatest Warner-Bratzler shear force (5.48 ± 0.12 kg) and matched for growth characteristics were chosen for the analyses of caspase 3 activity from the same 40 steers as experiment 2.
Caspase 3 Activity Assay
Caspase activity assay was performed using the EnzChek Caspase-3 assay kit 1 (Molecular Probes, Invitrogen, Carlsbad, CA). Frozen muscle samples (0.1 g) were homogenized in 500 µL of extraction buffer containing 10 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 1.0 mM EDTA, and 0.0001% Triton X-100. Samples were then frozen overnight at –20°C. Samples were thawed and centrifuged at 12,000 x g for 5 min. The assay was then carried out according to the kit protocol. Briefly, supernatant (50 µL) of each muscle homogenate was added to an individual well of a 96-well microfluorescent plate and incubated with or without 1 µL of Ac-DEVD-CHO inhibitor for 10 min at room temperature. After incubation, 50 µL of the 2 x working substrate (10 µM Z-Devd-AMC) were added to each well and further incubated for 30 min. Fluorescence was measured at 342 nm excitation and 441 nm emission using a spectrofluorometer (Germini XS, Molecular Devices, Union City, CA). Caspase 3 activity was expressed as micromoles of substrate cleaved per gram of muscle per minute.
Immunoblotting Analyses
Frozen muscle samples (0.1 g) were homogenized in 500 µL of extraction buffer containing 20 mM Tris-HCl (pH 7.4 at 4°C), 2% SDS, 1% Triton X-100, 5.0 mM EDTA, 5.0 mM EGTA, 1 mM DTT, 100 mM NaF, 2 mM sodium vanadate, 0.5 mM phenylmethylsufonyl fluoride, 10 µL/mL of leupeptin, and 10 µL/mL of pepstatin. Supernatant of each muscle homogenate was mixed with an equal amount of sample loading buffer containing 150 mM Tris-HCl (pH 6.8), 20% glycerol, 2 mM 2-mercaptoethanol, and 0.004% (wt/vol) bromophenol blue and boiled for 3 min.
The SDS-PAGE separation was performed using a resolving gel containing 12% (wt/vol) acrylamide/bisacrylamide (29:1), 375 mM Tris-HCl (pH 8.8), 0.1% SDS, 0.04% ammonium persulfate, and 0.028% TEMED, and a stacking gel containing 4% (wt/vol) acrylamide/bisacrylamide (29:1), 330 mM Tris-HCl (pH 6.8), 0.04% ammonium persulfate, and 0.028% TEMED. After electrophoresis, proteins on the gel were transferred to a nitro-cellulose membrane (Biorad, Hercules, CA) in a transfer tank system using a transfer buffer containing 20 mM Tris-base, 190 mM glycine, 0.1% SDS, and 20% ethanol.
Membranes were incubated in a blocking solution consisting of 5% nonfat dry milk in TBS/T [0.1% Tween-20, 50 mM Tris-HCl (pH 7.6), and 150 mM NaCl] for 1 h. Then, the membranes were incubated overnight in a rabbit anti-caspase 3 antibody (Cell Signaling Technology, Beverly, MA) with 1:1,000 dilution (vol/vol) or a monoclonal anti-calpastatin antibody (Affinity Bioreagents, Golden, CO) with 1:2,000 dilution (vol/vol) in TBS/T with 2% nonfat dry milk. At the end of the primary antibody incubation, the membranes were washed 3 times for 5 min each with 20 mL of TBS/T. After that, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody against rabbit IgG (1:2,000, vol/vol; Amersham Bioscience, Piscataway, NJ) for 1 h in TBS/T with gentle agitation. After three 5-min washes, the membranes were visualized using ECL Western blotting reagents (Amersham Bioscience) and exposure to film (MR, Kodak, Rochester, NY; Zhu et al., 2006).
Statistical Analysis
Experiment 1 data were analyzed using the MIXED (mixed models) procedure (SAS Inst. Inc., Cary, NC). Animal was used as the random effect because these animals were randomly selected from a group of 15 steers slaughtered at the University of Wyoming Meat Laboratory. Time was used as the fixed effect because the sampling time points were predetermined. Individual animal was considered the experimental unit. Experiment 2 and 3 data were analyzed as a completely randomized design using PROC GLM of SAS. Individual animal was considered as the experimental unit. Differences in mean values were compared by the LSD comparison, and least squares means ± SE were reported. Statistical significance was considered as P < 0.05, and trends were considered at P < 0.10.
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RESULTS AND DISCUSSION
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Proteolysis during the postmortem period is important for beef tenderness. Cathepsins and calpains/calpastatin system may be involved in postmortem proteolysis (Uytterhaegen et al., 1994; Koohmaraie, 1996). Current data supports the role of calpain/calpastatin system as the main participant in the postmortem tenderization (Koohmaraie et al., 1988; Uytterhaegen et al., 1994; Koohmaraie, 1996). However, calpain and calpastatin cannot explain all the variability in tenderness (Morgan et al., 1993; Koohmaraie, 1995). Koohmaraie (1995) attributed 46% of the variability in tenderness to genetic factors and credited the remaining 54% to environmental influences. Koohmaraie (1995) goes on to explain that 30% of the variation in beef tenderness is due to calpastatin, the specific inhibitor of calpains. The difference in postmortem tenderization cannot be fully explained by the variation in calpain/calpastatin system (Morgan et al., 1993). Therefore, it is possible that other proteolytic systems are active in postmortem muscle. Caspase 3 has been investigated in mouse (Condorelli et al., 2001) and pig (Kemp et al., 2006a,b) skeletal muscle, but has not been examined in postmortem beef muscle.
Caspases are cysteine proteinases which function in apoptosis or programmed cell death and exist as proproteins in muscle (Suzuki et al., 2001; Du et al., 2004; Fuentes-Prior and Salvesen, 2004; Herrera-Mendez et al., 2006). These proteinases function to cleave proteins at specific aspartic acid residues after activation, which requires cleavage of the pro-domain and dimerization (Sentandreu et al., 2002; Fuentes-Prior and Salvesen, 2004; Herrera-Mendez et al., 2006). Caspases can be classified into cytokine activators that function in inflammation, apoptotic initiators, and apoptotic effectors (Fuentes-Prior and Salvesen, 2004; Herrera-Mendez et al., 2006). Apoptosis is initiated by a caspase cascade involving initiator caspases 8, 9, or 10 (Fuentes-Prior and Salvesen, 2004). Caspase 3 is an effector caspase that is activated by other caspases and is responsible to execute most protein hydrolysis tasks. It has been documented to cleave myofibrillar proteins in muscle catabolic conditions (Du et al., 2004).
In skeletal muscle, caspase-3 exists as its pro-form, which is inactive. Pro-caspase 3 is activated by cleavage into a 14 kD caspase 3 that is active (Turpin et al., 2006). Therefore, if caspase 3 contributes to postmortem proteolysis, it must be cleaved into a 14-kD active form during postmortem. To show whether there was a 14-kD active form of caspase 3 present in beef muscle, we conducted immunoblotting analyses using a caspase 3 antibody that recognized both the pro- and active form of caspase 3. No active caspase 3 was detected in LT up to 7 d postmortem (Figure 1). Because beef longissimus muscle is mainly composed from type IIa muscle fibers (Underwood et al., 2007b), to test whether a muscle mainly composed of type I fibers behaves differently, we further analyzed caspase 3 in sternomandibularis muscle. Again, no active form of caspase 3 was detected (Figure 2). To confirm that our failure to detect 14 kD caspase 3 was not due to our technical limitation, a positive control that contains pro- and activated caspase 3 (Cell Signaling Technology, Beverly, MA) and a negative control that only contains pro-caspase 3 (Cell Signaling Technology) were loaded into gels together with muscle samples. Beef muscle samples at various postmortem stages only contain pro-caspase 3, which was also detected in the negative control (Figures 1 and 2). The active caspase 3 was detected in the positive control but absent in beef muscle samples (Figures 1 and 2). These results clearly showed that pro-caspase 3 was not cleaved into active caspase 3 at detectable quantities in postmortem muscle. However, immunoblotting is not a very sensitive method for detecting small amounts of low molecular weight proteins. Therefore, there is a chance that small amount of active caspase 3 exists in muscle samples that immunoblotting analyses fail to detect. To solve this problem, we further assessed the caspase 3 activity.
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Figure 1. Immunoblots of caspase 3 in beef longissimus thoracis muscle at 0, 0.25, 1, 3, 24, 72, and 240 h postmortem. Two representative immunoblots are shown. Positive control: Jurkat cell extract treated with cytochrome C; negative control: untreated Jurkat cells.
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Figure 2. Immunoblots of caspase 3 in beef sternomandibularis muscle at 0, 0.25, 1, 3, 24, 72, and 240 h postmortem. Two representative immunoblots are shown. Positive control: Jurkat cell extract treated with cytochrome C; negative control: untreated Jurkat cells.
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There was no significant activation of caspase 3 in LT muscle at postmortem (Figure 3). Caspase 3 activity at 0, 0.25, 1, 3, and 24 h was not changed, whereas caspase 3 activity was decreased at 72 (P = 0.05) and 240 h (P = 0.02) postmortem when compared with 0 h (Figure 3). The lack of any significant increase in caspase 3 activity during the postmortem stage following exsanguination shows this enzyme is not activated during the postmortem period, indicating that caspase 3 is unlikely to play a significant role in postmortem tenderization. These data are different from a previous report in pigs, which found that caspase 3 was activated in postmortem porcine skeletal muscle (Kemp et al., 2006b).
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Figure 3. Caspase 3 activity in beef longissimus dorsi muscle at 0, 0.25, 1, 3, 24, 72, and 240 h postmortem. Results are presented as means ± SE. *Values differ from the initial (0 h) time point (P < 0.05).
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To further evaluate the possibility of caspase 3 in postmortem proteolysis, we analyzed the link between caspase 3 activity and beef tenderness. Because beef tenderness is affected by numerous factors, such as genetics, age, and nutritional condition of cattle (Morgan et al., 1993; Koohmaraie, 1995), we used a group of cattle that had the same genetic background, age, and nutritional management to reduce tenderness variations due to the previous factors. We selected 5 steers with a low mean Warner-Bratzler shear force of 3.77 ± 0.20 kg and another 5 steers with a high mean shear force of 4.44 ± 0.18 kg (Underwood et al., 2007a). The low shear force group had less muscle percentage in the carcass as shown by smaller LT area size of 70.3 ± 1.5 cm2 compared with 81.4 ± 3.4 cm2 for high shear force group (P < 0.05). In addition, lighter LT weights (1.63 ± 0.03 kg versus 1.80 ± 0.05 kg, P < 0.05) and semitendinosus muscle weights (0.52 ± 0.03 versus 0.60 ± 0.04, P < 0.05) were observed in the low shear force group. We measured calpastatin using immunoblotting in these animals as calpastatin, the specific inhibitor of calpains, has been shown to function in muscle growth as well as postmortem proteolysis (Goll et al., 2003). We observed an increased calpastatin level (P = 0.05) in the high shear force group of experiment 2 when compared with the low shear force group (Figures 4 and 5). This illustrates the increased muscle growth and decreased tenderness of the high shear force cattle might be related to calpastatin levels. The caspase 3 assay surprisingly showed a lower (P = 0.05) caspase 3 activity in the low shear force group compared with high shear force group (Figure 4). Being that the cattle in experiment 2 showed a difference in muscle growth and tenderness that was related to calpastatin levels in these animals we analyzed another group of cattle. This group of animals (experiment 3) had a high shear force group exhibiting a Warner-Bratzler shear force of 5.48 ± 0.12 kg and low shear force group having a Warner-Bratzler shear force of 3.60 ± 0.12 kg (P = 0.0001, Figure 6). These animals, however, exhibited no differences in growth as shown by similar LT area, LT weights, and ST weights (P 
0.42, Table 1). Additionally this group of cattle had similar carcass composition as estimated by the 9-10-11th rib dissections (P 0.59, Table 1). The caspase 3 assay for experiment 3 showed no difference (P = 0.83, Figure 6) between the low and high shear force groups selected for similar growth characteristics. This suggests that caspase 3 activity at slaughter was not correlated with beef tenderness, further indicating that caspase 3 is unlikely contributing to the postmortem tenderization of beef muscle. Nevertheless, the increased caspase 3 activity showed an association with measures of muscle growth in these cross-bred steers, which warrants further studies.
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Figure 4. (A) Warner-Bratzler shear force of longissimus thoracis muscle from low- and high-shear force beef cattle. (B) Caspase 3 activity in longissimus thoracis muscle of low- and high-shear force beef cattle. Results are presented as means ± SE. *Values differ between low- and high-shear force (P < 0.05).
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Figure 5. Immunoblot of calpastatin in longissimus thoracis muscle from low- and high-shear force beef cattle. *Values differ between low- and high-shear force (P < 0.05). GAPDH = glyceraldehyde 3-phosphate dehydrogenase.
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Figure 6. (A) Warner-Bratzler shear force of longissimus thoracis muscle from low- and high-shear force beef cattle matched for growth characteristics. (B) Caspase 3 activity in longissimus thoracis muscle of low- and high-shear force beef cattle matched for growth characteristics. Results are presented as means ± SE. *Values differ between low- and high-shear force (P < 0.05).
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Table 1. Live BW and carcass characteristics of low- and high-shear force steers matched for growth characteristics
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In summary, our results show that there is caspase 3 activity in beef muscle immediately after slaughter and during the postmortem stage, but shows no significant increase in activity at postmortem time points measured. Also, we did not detect the activation of caspase 3 in the LT and sternomandibularis muscles of beef carcasses using immunoblotting. In addition, caspase 3 activity was not correlated with Warner-Bratzler shear force of beef LT. Therefore, caspase 3 is unlikely to participate significantly in the proteolysis of postmortem beef muscle.
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Footnotes
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1 This work was supported by Research Initiative Grants 2007-35203-18065 and 2006-55618-16914 from the USDA Cooperative State Research, Education and Extension Service. 
2 Corresponding author: mindu{at}uwyo.edu
Received for publication August 30, 2007.
Accepted for publication December 10, 2007.
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LITERATURE CITED
|
|---|
Condorelli, G., R. Roncarati, J. Ross Jr., A. Pisani, G. Stassi, M. Todaro, S. Trocha, A. Drusco, Y. Gu, M. A. Russo, G. Frati, S. P. Jones, D. J. Lefer, C. Napoli, and C. M. Croce. 2001. Heart-targeted overexpression of caspase3 in mice increases infarct size and depresses cardiac function. Proc. Natl. Acad. Sci. USA 98:9977–9982.[Abstract/Free Full Text]
Du, J., X. N. Wang, C. Miereles, J. L. Bailey, R. Debigare, B. Zheng, S. R. Price, and W. E. Mitch. 2004. Activation of caspase-3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions. J. Clin. Invest. 113:115–123.[CrossRef][Medline]
Etherington, D. J., M. A. J. Taylor, and E. Dransfield. 1987. Conditioning of meat from different species - relationship between tenderizing and the levels of cathepsin-B, cathepsin-L, calpain-I, calpain-II and β-glucuronidase. Meat Sci. 20:1–18.[CrossRef]
Fuentes-Prior, P., and G. S. Salvesen. 2004. The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem. J. 384:201–232.[CrossRef][Medline]
Goll, D. E., Y. Otsuka, P. A. Nagainis, J. D. Shannon, S. K. Sathe, and M. Muguruma. 1983. Role of muscle proteinases in maintenance of muscle integrity and mass. J. Food Biochem. 7:137–177.[CrossRef]
Goll, D. E., V. F. Thompson, H. Li, W. Wei, and J. Cong. 2003. The calpain system. Physiol. Rev. 83:731–801.[Abstract/Free Full Text]
Hankins, O. G., and P. F. Howe. 1946. Estimation of the composition of beef carcasses and cuts. In USDA (ed.) Technical Bulletin 926. USDA, Washington, DC.
Herrera-Mendez, C. H., S. Becila, A. Boudjellal, and A. Ouali. 2006. Meat ageing: Reconsideration of the current concept. Trends Food Sci. Technol. 17:394–405.[CrossRef]
Kemp, C. M., R. G. Bardsley, and T. Parr. 2006a. Changes in caspase activity during the postmortem conditioning period and its relationship to shear force in porcine longissimus muscle. J. Anim. Sci. 84:2841–2846.[Abstract/Free Full Text]
Kemp, C. M., T. Parr, R. G. Bardsley, and P. J. Buttery. 2006b. Comparison of the relative expression of caspase isoforms in different porcine skeletal muscles. Meat Sci. 73:426–431.[CrossRef]
Koohmaraie, M. 1995. The biological basis of meat tenderness and potential genetic approaches for its control and prediction. Pages 69–76 in Recip. Meat Conf., San Antonio, TX.
Koohmaraie, M. 1996. Biochemical factors regulating the toughening and tenderization processes of meat. Meat Sci. 43:S193–S201.[CrossRef]
Koohmaraie, M., A. S. Babiker, R. A. Merkel, and T. R. Dutson. 1988. Role of Ca++-dependent proteases and lysosomal-enzymes in postmortem changes in bovine skeletal muscle. J. Food Sci. 53:1253–1257.[CrossRef]
Koohmaraie, M., G. Whipple, D. H. Kretchmar, J. D. Crouse, and H. J. Mersmann. 1991. Postmortem proteolysis in longissimus muscle from beef, lamb and pork carcasses. J. Anim. Sci. 69:617–624.[Abstract]
Lee, S. W., G. Dai, Z. Hu, X. Wang, J. Du, and W. E. Mitch. 2004. Regulation of muscle protein degradation: Coordinated control of apoptotic and ubiquitin-proteasome systems by phosphatidylinositol 3 kinase. J. Am. Soc. Nephrol. 15:1537–1545.[Abstract/Free Full Text]
Morgan, J. B., T. L. Wheeler, M. Koohmaraie, J. W. Savell, and J. D. Crouse. 1993. Meat tenderness and the calpain proteolytic system in longissimus muscle of young bulls and steers. J. Anim. Sci. 71:1471–1476.[Abstract]
Sentandreu, M. A., G. Coulis, and A. Ouali. 2002. Role of muscle endopeptidases and their inhibitors in meat tenderness. Trends Food Sci. Technol. 13:400–421.[CrossRef]
Suzuki, K., S. Kostin, V. Person, A. Elsasser, and J. Schaper. 2001. Time course of the apoptotic cascade and effects of caspase inhibitors in adult rat ventricular cardiomyocytes. J. Mol. Cell. Cardiol. 33:983–994.[CrossRef][Medline]
Turpin, S. M., G. I. Lancaster, I. Darby, M. A. Febbraio, and M. J. Watt. 2006. Apoptosis in skeletal muscle myotubes is induced by ceramides and is positively related to insulin resistance. Am. J. Physiol. Endocrinol. Metab. 291:E1341–E1350.[Abstract/Free Full Text]
Underwood, K. R., W. J. Means, M. J. Zhu, S. P. Ford, B. W. Hess, and M. Du. 2007a. AMP-activated protein kinase is negatively associated with intramuscular fat content in longissimus dorsi muscle of beef cattle. Meat Sci. In press.
Underwood, K. R., J. Tong, M. J. Zhu, Q. W. Shen, W. J. Means, S. P. Ford, S. I. Paisley, B. W. Hess, and M. Du. 2007b. Relationship between kinase phosphorylation, muscle fiber typing and glycogen accumulation in longissimus dorsi muscle of beef cattle with high and low intramuscular fat. J. Agric. Food Chem. 55:9698–9703.[CrossRef][Medline]
USDA. 1997. United States standards for grades of carcass beef. Washington, DC.
Uytterhaegen, L., E. Claeys, and D. Demeyer. 1994. Effects of exogenous protease effectors on beef tenderness development and myofibrillar degradation and solubility. J. Anim. Sci. 72:1209–1223.[Abstract]
Zhu, M. J., S. P. Ford, W. J. Means, B. W. Hess, P. W. Nathanielsz, and M. Du. 2006. Maternal nutrient restriction affects properties of skeletal muscle in offspring. J. Physiol. 575:241–250.[Abstract/Free Full Text]
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Abstract
INTRODUCTION
